review article alginate biosynthesis in azotobacter...

Review ArticleAlginate Biosynthesis in Azotobacter vinelandiiOverview of Molecular Mechanisms in Connection withthe Oxygen Availability

Ivette Pacheco-Leyva Felipe Guevara Pezoa and Alvaro Diacuteaz-Barrera

Escuela de Ingenierıa Bioquımica Pontificia Universidad Catolica de Valparaıso Avenida Brasil 2147 Casilla 4059 Valparaıso Chile

Correspondence should be addressed to Alvaro Dıaz-Barrera alvarodiazucvcl

Received 28 August 2015 Revised 11 January 2016 Accepted 7 February 2016

Academic Editor Mukund Adsul

Copyright copy 2016 Ivette Pacheco-Leyva et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The Gram-negative bacterium Azotobacter vinelandii can synthetize the biopolymer alginate that has material propertiesappropriate for plenty of applications in industry as well as in medicine In order to settle the foundation for improving alginateproduction without compromising its quality a better understanding of the polymer biosynthesis and the mechanism of regulationduring fermentation processes is necessary This knowledge is crucial for the development of novel production strategies Herewe highlight the key aspects of alginate biosynthesis that can lead to producing an alginate with specific material properties withparticular focus on the role of oxygen availability linked with the molecular mechanisms involved in the alginate production

1 Introduction

Increasing research on the mechanisms of synthesis and bio-chemical properties of biopolymers such as polysaccharidesled to improving production process and new applications indiverse areas mainly in food and pharmaceutical industries[1] One of the main advantages for the use of biopolymers isits degradability making them a renewable product optionHowever the high costs of biopolymer production are still amajor drawback for a widespread industrial application [2]

A particular linear polysaccharide with broad growinginterest is alginate which is a structural component of thebrown marine algae and the cell wall of bacteria belongingto the Pseudomonas and Azotobacter genera [3ndash5] The prop-erties of alginates in solution largely depend on four factors(a) its monomer chemical composition (120573-D-mannuronicacid (M-residues) and its epimer 120572-L-guluronic acid (G-residues)) (b) the sequence pattern of the monomers (c)the molecular weight (MW) of the resulting polysaccharidechain and (d) modifications of the polymer (acetylationdegree) [6 7]

However algal alginates are complexmixtures containingpolysaccharides with a wide range of MW and ratios of

M G Hence alginates with specific defined M G ratios ora constant range of MW cannot be easily obtained fromparticular algae species due to intrinsic environmental cul-ture conditions thus limiting their use in the pharmaceuticaland chemical industries (more details in Section 2) For thisreason the bioprocesses research area has become interestedin developing strategies to produce alginates with particularmolecular characteristics throughmicrobial alginate produc-tion In contrast to algal alginates microbial alginates presentexclusive M-residue acetylation controlled M G ratios andspecific MW under specific growth conditions [8ndash10] Anonpathogenic bacterium able to produce alginate with highproduction yields in bioreactors is Azotobacter vinelandiiYet the complex regulatory pathways controlling the alginatebiosynthesis and material properties in response to externalenvironmental clues remain still unknown despite someefforts in trying to gain new insights into gene expressionpatterns under different culturing conditions in A vinelandiicultures [9 11ndash13]

In this review we present an up-to-date biosyntheticoverview of microbial alginate biosynthesis fromAzotobactervinelandii and the perspectives for production processimprovement based on a better understanding on the

Hindawi Publishing CorporationInternational Journal of Polymer ScienceVolume 2016 Article ID 2062360 12 pageshttpdxdoiorg10115520162062360

2 International Journal of Polymer Science

2000

1500

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1975 1995 2015

Year(a)

1657 biochemistry genetics and molecular biology1093 medicine1083 materials science1057 chemical engineering943 chemistry900 engineering769 agricultural and biological sciences748 immunology and microbiology604 pharmacology toxicology and pharmaceutics1147 others

Total = 43600

(b)

Figure 1 (a) Number of publications indexed in Scopus database(August 2015) using keyword alginate (minus) in title abstract orkeywords (b) Percentage of theword alginatedistributed in differentsubject areas

molecular mechanism underlying polymer biosynthesis inrelationship with the oxygen availability during the fermen-tation process

2 Alginate Structure Chemical Structureand Applications

Over the past 40 years a growing interest in the use of alginatehas been observed including different areas ranging fromgenetics to pharmaceutics (Figure 1)

Alginate has been placed as the second biopolymerderived from seaweeds with greater demand in the

Table 1 Summary of biotechnological and pharmaceutical applica-tions of alginates based on their molecular weights

Application of alginateMolecularweight(kDa)

Reference

Delivery of bioactive compoundsAntioxidantIn vivo tissue scaffoldsAntibacterialDietary supplementCell immobilization

asymp15ndash120 [60ndash66]

Food stabilizer and preservingagentMicroencapsulation and storagestabilityAntibacterialBioremediationWound healing

asymp120ndash290 [62 63 67ndash76]

Modulation of enzymatic activityExtended-release tabletcompound

500ndash941 [71 77]

hydrocolloidsrsquo industry [14] Currently the only economicway to obtain commercial alginate used for most applicationsis through its extraction frommarine algae the cost of whichranges between US$ 2 and 20kg and with a total marketvalue of around US$ 339 million [14] Furthermore alginatesof very high purity are used in the pharmaceutical industrywhere they are sold for up to US$ 3200kg

Since alginate is a biodegradable and a biocompatiblepolysaccharide it presents a panoply of food pharmaceu-tical and biotechnological applications (Figure 1(b)) In thefood and pharmaceutical industries alginate is mainly usedas a stabilizing thickening or gel-film-forming agent [615ndash17] Table 1 in medicine it is used as wound healingmaterial [18] as part of medical treatments [19 20] or asdietary fiber supplements [21 22] Alginate showed potentialbeneficial physiological effects in the gastrointestinal tract[23] Moreover hydrogel-alginates are being investigated inbiotechnology as drug delivery agents as cell encapsulationmaterial and as scaffold material in tissue engineering [24]

Alginate is the main structural component of brownmarine algae (Laminaria andMacrocystis) representing about32 of dry biomass [25] consisting in variable amounts ofM- G- and MG-residues linked by 1rarr4 glycosidic bonds[7] On the other hand alginates produced by bacteria aresubmitted to esterification with O-acetyl groups at the O-2 andor O-3 of the M-residues [26] where the majority oftheM-residues aremono-O-acetylated and infrequentlywith23-di-acetylated [27] (Figure 2) Because the monomericchemical structure of bacterial alginate and the sequencelength determine the mechanical properties of the alginatesone of the aims of different investigations is the possibility ofmanipulating the composition alginates for specific applica-tions have been intensively investigated [28 29]

The obligate aerobe bacterium Azotobacter vinelandiiproduces alginate that acts as a diffusion barrier for nutrientsand oxygen [30 31] It was reported as a bacterium with a

International Journal of Polymer Science 3

O

OO

OO

OO

OO

O OO

O

OH

OH

OHOH

HO

OO

M M M

OH

OHG G

OHOH

OH

G

minusOOC

minusOOC

minusOOC

minusOOC

minusOOC

minusOOCH3C

CH3

CH3

C=O

C=OC=O

Figure 2 Representation of the chemical structure from acetylated alginates produced byAzotobacter vinelandii bacterium [28]Mannuronic(M) and guluronic (G) acid residues are represented in the alginate chain

highest respiratory rate [32] implying that it adjusts oxygenconsumption rates in order tomaintain low levels of cytoplas-mic oxygen and in this way permitting the oxygen-sensitiveenzymes to be active like nitrogenase which is responsiblefor fixing nitrogen [30 32]

A vinelandii under limitation of carbon source or byinduction forms cysts that are more resistant to desiccationand is mainly composed of alginate [33 34] It also accu-mulates the intracellular polyester poly-120573-hydroxybutyrate(PHB) as a reserve carbon and energy source [35 36]

Consequently an increased knowledge about the molec-ular mechanism involved in alginate biosynthesis will becrucial for the development of novel strategies to improve theproduction of alginates with defined characteristics tailoredfor specific applications

3 The Biosynthetic-SecretoryRoute of Alginate Production inAzotobacter vinelandii

Microbial polysaccharides have distinct biological functionsas intracellular storage as envelope or as extracellular poly-mers [37] Microbial alginate is an extracellular polysaccha-ride as xanthan cellulose and sphingan among others andthey differ in their biosynthetic pathways routes (recentlyreviewed in Schmid et al 2015 [37]) Moreover alginate issecreted trough a secretion system shared among the Gram-negative bacteria [38]

The alginate biosynthesis in bacteria Azotobacter resultsfrom a complex regulatory network of proteins similar toPseudomonas genera [6 28 39]

All of the steps involved in the conversion of cen-tral sugar metabolites into the alginate precursor in Avinelandii have been previously identified and character-ized [6 40] The alginate precursor GDP-mannuronic acidis synthesized from fructose-6-phosphate to mannose-6-phosphate by the bifunctional enzyme phosphomannoseisomerase (PMI)guanosine-diphosphomannose pyrophos-phorylase (GMP) designated as AlgA encoded by the algAgene A phosphomannomutase (AlgC) directly converts themannose-6-phosphate into mannose-1-phosphate which isin turn converted into GDP-mannose by the AlgA enzymeGDP-mannose is oxidized toGDP-mannuronic acid byGDP-mannose dehydrogenase (AlgD encoded by algD gene)Because the intracellular levels of GDP-mannose are high andbecause it is used in different pathways it has been proposed

as the limiting step of alginate biosynthesis in P aeruginosa[41]

After the production of the polymer precursor GDP-mannuronic acid precursor its polymerization and transportacross the cytoplasmic membrane is carried out by proteinspresumably integrating a cytoplasmic membrane complex(polymerase complex)The core of the polymerase complex iscomposed of the glycosyltransferase Alg8 protein and Alg44protein [42ndash44] Furthermore the protein AlgK is thought tostabilize the polymerase complex by interacting with Alg44[43] Highlighting the important role of this protein alginatepolymerization does not occur in the absence of algK [42 45]

The polymannuronate polysaccharide resulting frompolymerization and then translocation to the A vinelandiiperiplasm is composed of M-residues which can then befurthermodified during its passage across the periplasm [43]These modifications consist in acetylation epimerizationand degradation of the M-residues More specifically thepolymannuronic molecule undergoes anO-acetylase modifi-cation which is catalyzed by an acetylase enzymatic complexcomposed of AlgI AlgV (AlgJ in P aeruginosa) AlgF andAlgX proteins [46ndash48] While M-residue O-acetylation doesnot occur frequently in alginate some may be acetylatedO-acetylated M-residues will therefore be protected fromepimerization [26] because only nonacetylated M-residuescan be epimerized to G-residues by the AlgG epimerase [42]so alginates with a relatively high degree of acetylation displaya lower degree of epimerization [27]

Alginate depolymerization occurs at the 4-O-glycosidicbond via 120573-elimination by alginate lyases which have beenthe subject of a recent review [28]TheAzotobacter vinelandiigenome encodes six enzymes with alginate lyase activity [31]the alginate lyase AlgL [49] the bifunctional mannuronanC-5 epimerase and alginate lyase AlgE7 [50] the threeAlyA(1ndash3) lyases [51] and an exolyase AlyB that is stilluncharacterized [28]

Some of the nonacetylated M-residues are then epimer-ized toG-residues by the bifunctional AlgG epimerase whichconverts poly(120573-D-mannuronate) to 120572-L-guluronate In Paeruginosa AlgG is also part of the periplasmic proteincomplex that serves as a scaffold for leading the newlyformed alginate polymer through the periplasmic spaceto the outer membrane secretin AlgE porin (AlgJ in Avinelandii) [52] A scaffold complex helps to transport therecently modified polysaccharide throughout the periplasmtowardsAlgE before secretion to the extracellularmilieuThis


OM

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Synthesis and polymerization of precursors Periplasmic and extracellular modifications

AlyB

E1 E2E6

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AlyA1AlgF

AlgV AlgGAlgAlgGAlgGggGgggGg

Algl Alg8AAAAAA ggg8888AA gg88

Acetyl donor

Acetyl group

OM outer membrane

PP

PGDP

P GDP-mannoseGDP-mannuronic acidMannose-1-phosphate

Mannose-6-phosphateFructose-6-phosphate

Mannuronate residueGuluronate residueUnknown function

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gAlg44

AlgX

AlgJAAAlggAAA gg AAAlgKlAAlggKKKAAAAA

AlgL

AlgK

Alg44AlgG

AlglAAAAlglgggllA gg Alg8

c-di-GMP

GMP + Pi

Ca2+

Ca2+

Ca2+Ca2+ Ca2+

Figure 3 Schematic representation of the alginate biosynthetic steps in Azotobacter vinelandii from evidence-based protein-proteininteraction in P aeruginosa [28 42 43] The biosynthetic alginate pathway is represented as two complementary stages on the left thesynthesis of the substrate precursor (GDP-mannuronic acid) and its following polymerization including transfer from cytoplasm on theright the modification (periplasmic and extracellular) of the nascent polymer as well as the export through the outer membrane of thepolymer

complex is thought to be composed of AlgG AlgK and AlgXproteins and possibly AlgL [40 42 43 52] The exportedpolysaccharide could be then epimerized by seven extracel-lular Ca2+-dependent epimerases (AlgE1ndash7) [53] Based onthese evidences Figure 3 shows a schematic representation ofthe alginate biosynthetic steps in A vinelandii

4 Genetic Regulation of Alginate Biosynthesisin Azotobacter vinelandii

In Azotobacter vinelandii the alginate biosynthetic genecluster is arranged as an operon (Figure 4) containing genescoding for enzymes involved in the synthesis of the alginateprecursors as well as those involved in its polymerizationdegradation acetylation epimerization and secretion Theavailability of the complete genome sequence of A vinelandii[31] also contributes to the better knowledge of this organism

Several promoters controlling alginate gene cluster tran-scription have been described algDp1 (120590D promoter) algDp2(AlgU 120590E dependent promoter) and algDp3 promoters alllocated upstream of algD [54 55] alg8p promoter upstreamof alg8 [44] and a promoter for sigma 70 located upstream

of algG [49] In addition two putative promoters algCp1 andalgCp2 are situated upstream of algC gene (Figure 4) [56]

The alginate biosynthetic gene cluster expression is con-trolled by algUmucABCD gene cluster where algU encodesthe alternative sigma 120590E factor (AlgU) essential for alginateproduction [57] Moreover AlgU is responsible for transcrip-tion driven by the algCp1 and algDp2 promoters (Figure 5)but it does not control the algL or the algA genes as describedfor P aeruginosa [55]

The MucA and MucC proteins negatively regulate algi-nate production acting as anti-120590E factors [54] MucArepresses AlgU protein activity thus suppressing algD tran-scription from the algDp2 promoter In contrast algU genetranscription is autoregulated by AlgU interaction and acti-vation of its 120590D promoter locus (algUp2) (Figure 5) [54]

Additionally expression of the algD promoters is con-trolled by the global two-component system GacSGacAwhich is conserved among Gram-negative bacteria [58] TheGacSGacA system controls alginate biosynthesis [58] whereGacS controls the expression of algD from its three promoters[58] Accordingly mutations in gacS and gacA significantlyreduce the algD transcript levels [58] GacA not only is apositive regulator of the biosynthesis of alginate andPHB [58]


algD alg8 alg44 algK algJ algG algX algL algl algV algF algA algC

algE7algE5algE6algE4

algE1algE2algE3alyA2

alyA3 alyBalyA1

Figure 4 Genetic structure genes involved in alginate biosynthesis and modification in Azotobacter vinelandii Gene operon for alginatebiosynthesis algD-A and algC gene is transcribed separately alyA1ndash3 and alyB alginate lyases encoding genes and algE1ndash7 the epimerasesgenes

OM

PGPe

ripla

sm

Alginate

IMCy

topl

asm

MucB

MucA

AlgU

GacS

GacA GacA

algCrpoS

algDrsmA

rsmrsmZ1rsmZ2

malgDalgU

120590D

120590S

ADP ATP

P

Cell wall stress

5998400UTR

Figure 5 Regulation of alginate biosynthetic genes in A vinelandii (modified according to reference [40]) Promoters are indicated asbanners mRNAs are indicated as dotted boxes solid lines indicate the reportedmechanism of regulation and dashed lines indicate unknownmechanism of gene regulation arrows indicate positive regulation and T-shaped bars indicate negative regulation OM outer membrane PGpeptidoglycan IM inner membrane See text for a more detailed description

but also regulates alginate biosynthesis through activation ofthe small regulatory RNAs Rsm (rsmZ1 and rsmZ2) TheseRNAs interact with the rsmA protein which binds algDmRNA and thus acts as a transcriptional repressor [59] TheA vinelandii genome encodes nine small RNAs belonging tothe Rsm posttranscriptional regulatory system (rsmZ1ndash7 andrsmY1-2) (Figure 5) [59]

Despite the great efforts to understand the alginate bio-synthetic gene regulation little is known about how cul-tivation conditions could modify gene transcription in Avinelandii

5 Alginate Production in Azotobactervinelandii Cultures The Balance of Alg8and AlgL by Oxygen Availability

The glycosyltransferase Alg8 protein belongs to the glycosyl-transferase type II family and is localized in the inner cellmembrane [78] The glycosyltransferase type II enzyme fam-ily catalyzes the transfer of glycosyl residues to an acceptormolecule during biosynthesis of polysaccharides such as thecellulose or chitin synthase [79]

In both Azotobacter vinelandii and Pseudomonas aerugi-nosa the alg8 gene encodes the Alg8 protein [44] In P aerug-inosa it has been demonstrated that by adding additionalcopies of alg8 it is possible to increase alginate productionby at least 10 times [80] suggesting that this protein mightbe involved in a rate-limiting step of alginate productionAs a consequence the possibility of manipulating Alg8 pro-tein levels in A vinelandii may be a valuable approach forincreased alginate production although this has not beingdone so far The attempts to reach high Alg8 protein levelswere by manipulating the alg8 gene expression via cultureconditions However it is important to note that alginateproduction in A vinelandii is a multienzymatic and complexprocess

Moreover the Alg44 protein acts as link between Alg8and the AlgJ alginate exporter protein [42 43] Since Alg44has a c-di-GMP intracellular binding domain it was sug-gested that this protein presents a regulatory role [81]although the c-di-GMP levels might not have an impactneither on Alg44 stability nor on its localization it still seemsto be required for the activation of Alg8 [42 43]

Interestingly in A vinelandii batch cultures controllingthe dissolved oxygen tension (DOT) at 1 present higher


Table 2 Molecular weight of alginate and relative gene expressionof alg8 and algL with respect to the 119902O2 variations

Specific oxygen uptake rate (mmol gminus1 hminus1) 1ndash5 5ndash10Alginate molecular weight (kDa) 500ndash1350 480ndash870algL gene expression (fold change) Until 15 05ndash15alg8 gene expression (fold change) Until 90 10ndash20Source [9 11ndash13]

levels of alg8 and alg44 gene expression when comparedwithcontrol cultures (5 DOT) [9] the authors suggested thatthis behavior can in turn enhance the MW of the alginateproduced under low DOT conditions Moreover in contin-uous cultures under non-nitrogen-fixation conditions at dif-ferent agitation rates (300 500 and 700 rpm) and differentsucrose concentration in the feed medium the highest algi-nateMW (obtained at 500 rpm) is correlated with the highestalg8 expression [12] suggesting that alg8 gene expression canbe modulated by not only oxygen availability but also carbonsource feed rate as well The oxygen availability here is per-ceived as the amount of oxygen needed for full oxidationof carbon source taking into account the oxygen transferrate as well as the DOT level in cultures [82] Meanwhile inchemostat cultures under nitrogen-fixation conditions oper-ated at a dilution rate of 007 hminus1 expression of both alg44and alg8 was affected by changes in agitation rate (400 500and 800 rpm) implying that the activity of both genes couldbe controlled by oxygen availability [13] Although the highestalginateMWwas obtained at 500 rpm this was not correlatedwith higher alg8 gene expression which was obtained at800 rpmThe differences between the two-chemostat cultureconditions might be explained by the activation of the nitro-genase protection machinery (non-nitrogen-fixation versusfixation) where the higher alginate MW have directly linkedto the alg8 gene expression under nonfixing conditions Thisnotion agrees with the fact that nitrogenase activity protectscells from oxygen thus fostering alginate production [30 83]Other possible explanation given is that the culture conditionmight activate the genes coding for alginate lyases furtherdiscussed in this review However more studies are neededespecially those involving gene expression and proteomicsprofiles during A vinelandii cultures in order to have a betterinsight of alginate polymerization step

A possible link among the low specific oxygen uptakerate (119902O

2

) the MW of the alginate synthesized and alg8 geneexpression was found [11] This work suggests that when the119902O2

value increases by double the MW of alginate decreases(about 16 times) while alg8 relative expression decreasesaround sixfold Moreover in cultures carried out in con-tinuous mode operated at dilution rate 008 hminus1 when the119902O2

value was 22mmol gminus1 hminus1 both the alginate MW andalg8 gene expression levels were higher than those obtainedin cultures in which the 119902O

2

value was double [11] The samecorrelation between low 119902O

2

value and highest alginate MWwas reported [12] where a slight increment of 1 in the 119902O

2

lead to a reduction in the MW of the alginate produced byA vinelandii (from 1200 to 500 kDa) Furthermore in this

condition the lyase-encoding gene algL increased its expres-sion by threefoldwhile alg8 expression decreased by ninefoldInterestingly for 119902O

2

values below 2mmol gminus1 hminus1 [12] orexceeding 5mmol gminus1 hminus1 [9 13] the changes in the alginateMW were not correlated with alg8 or algL gene expressionlevels Table 2 summarizes the major changes observed onboth the alginate MW and gene expression levels during thesmall increment values over the specific oxygen uptake rateof A vinelandii cultures

Furthermore theAzotobacter vinelandii genome encodessix enzymes with alginate lyase activity [31] the alginate lyaseAlgL [49] the bifunctional mannuronan C-5 epimerase andalginate lyase AlgE7 [50] and the three AlyA(1ndash3) lyases [51]

The AlyA1 AlyA2 and AlyA3 belong to the PL7 polysac-charide lyase family containing an alginate lyase modulelinked to three calcium-binding modules [28 51] AlyA1 andAlyA2 are more likely to be periplasmic (AlyA1 UniProtKB-M9YEJ6 AlyA2 UniProtKB-C1DHI8) whereas the AlyA3protein has secreted signal C-terminal domain (AlyA3UniProtKB-C1DQS5) which is needed for efficient germina-tion in A vinelandii [51] In chemostat cultures conductedat dilution rate of 007 hminus1 with agitation of 500 rpm highestalginate MW was reported [13] In this condition an incre-ment in the agitation rate (from 400 to 600 rpm) leads to anincrement in the lyase-encoding genes alyA1 algL and alyA2by twofold

The algGXLIVFA operon encodes the AlgL proteinresponsible for the periplasmic alginate lyase activity in Avinelandii Disruption of the algL gene generated a strain thatoverproduces alginate suggesting that this enzyme is impor-tant for alginate biosynthesis [84] Furthermore the increasein algL expression was not correlated with a decrease inalginate MW in chemostat cultures [12] However algL geneexpression pattern could also be affected by the 119902O

2

(manip-ulated by changes in the agitation rate) in chemostat Sup-porting this observation chemostat cultures also showed anincrease in algL gene expression (around eightfold) togetherwith higher MW alginate production [11 12] By using an Avinelandiimutant strain carrying algLWGmnonpolarmuta-tion [84] and culturing under 3 of DOT no alterations werefound in alginate lyase activity in culture broth comparingwith the wild-type strain However alginates with a highMWwere obtained [85] suggesting that the lower MW of thealginate correlates with the higher alginate lyase AlgL activity

In A vinelandii ATCC 9046 strain cultures carried outat 1 and 5 DOT the expression of higher alginate lyasegenes (algL alyA1 alyA2 alyA3 and algE7) correlated withthe lower DOT and with the higher MW alginate production[9] In these conditions (1DOT) the intracellular and extra-cellular lyase activities were lower comparing with culturesgrown at 5 DOT suggesting that dissolved oxygen affectedthe activity of the alginate lyases andor their gene expressionHowever the alginate lyase activity (intracellular and extra-cellular) seemed to be associated with the exponential phaseof the cultures where in the ATCC strain cultured themaxi-mum of alginate lyase activity was found in the prestationaryphase and dropping in the stationary phase [9 85]

As stated previously (Table 2) in cultures with 119902O2

between 2mmol gminus1 hminus1 and 5mmol gminus1 hminus1 [9 11ndash13] the


activity of intracellular lyases namely AlgL presented a basallevel which was not correlated with a rise in their genetranscriptional levels [9]This behavior per semay explain theobserved rise in alginate MW (Table 2) Even though theseobservations indicate that dissolved oxygen affects intra-cellular as well as extracellular alginate lyase activities it ispossible that different alginate lyases could be expressed atdifferent physiological states as suggested by the study ofAlyE3 which is essential for the efficient cyst germination inA vinelandii [51]

It is important to note that although the AlgL is localizedin the periplasm it has an N-terminal secretion signal (AlgLUniProtKB-O5219) suggesting that AlgL secretion can occurin response to diverse environmental stimuli (ie oxygenconcentration) This notion is supported by the observationthat AlgL extracellular activity is highly dependent on thedissolved oxygen and that the role of alginate lyase isrestricted to a postpolymerization step [9 85] Similarlythe alginate lyase AlyA3 also presents extracellular activitywhereas AlyA1 and AlyA2 appear to be periplasmic [51]These data strongly suggest that alginate lyase expression andextracellular activity occur in response to dissolved oxygenconcentrations Therefore a detailed analysis of dynamicvariations in expression levels and in enzymatic activitythroughout the culture is warranted to understand moredeeply the alginate polymerization process

In summary current evidence indicates that when valuesof 119902O

2

vary between 2 and 5mmol gminus1 hminus1 in cultures ofA vinelandii a rise in expression of algL together with adecrease in expression of alg8 correlates with a decrease inalginate MW (Table 2) As such this range of 119902O

2

could bea target in the development of strategies to manipulate thecharacteristics of alginates

51 Oxygen Sensing Mechanisms in Azotobacter vinelandiiCurrent evidences demonstrate that the oxygen transfer ratethe dissolved oxygen tension levels and the oxygen uptakerate affect alginate biosynthesis in A vinelandii cultures [8 912 13 36 40 86ndash89] Despite the importance of the oxygenand the intrinsic relationship with it no strong evidence ofthe molecular mechanism involved in sensing it during Avinelandii culturing is available as well as its further down-stream mechanism still being lacking In this section wediscuss that oxygen availability duringA vinelandii culturingis a key factor and we suggest a possible mechanism of action

In A vinelandii the mechanism involved in sensingoxygen availability remains to be fully investigated In bac-teria several oxygen sensing mechanisms exist Howeverthey can be clustered in two groups based on how thesignal is perceived One category can interact with externalenvironment while on the other hand the second categorysenses physiological changes resulting from variations in theexternal environment Nevertheless both sensing mecha-nisms operating together control directly the switch betweenaerobic and anaerobic metabolism [90] Among the oxygensensing mechanism the FNR ArcAB and ubiquinone-8(Q8) are well characterized in E coli [90]

In A vinelandii the absence of an Fnr-like proteinCydR overexpressing the120573-ketothiolase and acetoacetyl-coA

reductase [91] both enzymes catalyze the production of 120573-hydroxybutyryl-CoA which is the PHB precursor [40] Ithas been demonstrated that low aeration culture conditionsin A vinelandii cultures enhanced the metabolic flux frompyruvate towards acetyl-CoA This had an influence on theincrement on the metabolic flux towards PHB productionconcomitantly with the higher alginate production [8] sug-gesting that the aeration conditions could affect the alginateproduction by regulating possible gene targets of CydRSupporting this observation batch cultures of A vinelandiiOP mutant strain carried out at 600 rpm showed lowest 119902O

2

compared with wild-type strain (ATCC 9046) [92] The AvinelandiiOP strain contains an insertion element in the algUgene which in turn represses alginate synthesis [93] and it hasbeen suggested thatAlgU is required for cydR gene expression[94]

CydR controls the expression of cydAB operon thatencodes a cytochrome bd terminal oxidase and cydABgene expression correlates with the NADHubiquinoneoxidoreductase activity (NDHII) [91] In A vinelandiithe Na+-translocating NADHubiquinone oxidoreductases(Na+-NQR) are encoded in the nqr operon and it hadbeen linked to regulating negatively alginate production [95]Additionally A vinelandii genome contains genes linkedto NADHubiquinone oxidoreductases (NDH) the NDH-IItype and 13 genes encoding subunits of NDH-I type [95]The NADH oxidation in A vinelandii is mediated by twoNADHubiquinone oxidoreductases [96] and the fast NADHoxidation is linked to a fast quinone reduction The ubiC-Aoperon in A vinelandii is responsible for the transcription ofthe genes necessaries for Q8 biosynthesis [95] A mutation inthe intragenic region ubiA correlates with lower Q8 proteinlevels accompanied with an improvement in the alginateproduction but all the more with a higher expression ofbiosynthetic alginate genes algD algC and algA Moreoverthe Q8 protein seems to be responsible for at least 8 ofthe respiratory capacity in A vinelandii during low and highaeration cultures [95]

Interestingly in other bacteria as E coli the role ofquinones as a redox signal for the pathways involved in sens-ing oxygen and regulation of expression of genes involvedin oxidative and fermentative catabolism is well knownspecifically the ArcBA two-component system [97ndash99]

Figure 6 summarizes the plausible regulation of alg genesin A vinelandii via a signaling cascade activated by oxygenavailability On one hand the Na+NQR protein regulatesnegatively algD and algC gene targets while the ArcBA two-component system regulates algD and alg8 gene expressionunder oxygen availability When oxygen is limiting thesensor kinase ArcB autophosphorylates and then transphos-phorylates the regulator ArcA which activates algD alg8 andalg44 gene expression The autophosphorylation of ArcB isinhibited at higher oxygen concentrations by the accumula-tion of Q8 (oxidized form) In this sense in A vinelandii atight control of alg genes via a signaling cascade activated byoxygen availability may exist (Figure 6)

Although recently Flores et al 2015 [36] discussedmainly the influence of the oxygen on production of alginateduring A vinelandii cultures not much attention is paid


High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM

algD algD algDalg8 alg44

Na+NQRNADHdehydrogenase

NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+

Figure 6 Schematic representation of the possible gene regulation mechanism by oxygen in Azotobacter vinelandii Oxygen availability isdepicted in the figure as low O

2(left side) and high O

2(right side) Light red dotted boxes indicate the Na+-translocating NADHubiquinone

oxidoreductase (Na+NQR) that regulates negatively algD and algC gene targets although the exact mechanism of algD and algC generegulation at highO

2byNa+NQR is still unknownGray slashed boxes represent theArcBA two-component redox sensor under high oxygen

availability the autophosphorylation of ArcB (B blocks) is inhibited by oxidized quinones (Q8) ArcA (A blocks) in the nonphosphorylatedstate is unable to bind specifically to algD alg8 and alg44 gene targets Low oxygen causes a decrease in the level of oxidized quinones(Q8H2) allowing the autophosphorylation ofArcAArcA-P binds specifically to its target sites and coordinates the cellular response to oxygenavailability Arrows indicate positive regulation and T-shaped bars indicate negative regulation Flag-type boxes indicate genes described inthe figure Question mark indicates unknown gene regulation mechanism OM outer membrane PG peptidoglycan IM inner membrane

to which molecular pathways are involved during alginatebiosynthesis In our work we propose a possible mechanismof action of the oxygen availability during A vinelandiiculturing offering a new path to look at and in this waycontributing to the better knowledge of controlling bacterialalginates production

Despite the enormous efforts in understanding themicro-bial alginate biosynthesis under defined culture conditionsthere is still a way to go The decoding of the A vinelandiigenome has open the possibility to getting access to newinformation however no wide genetic screen studies duringalginate production have been reported yet So it will benecessarily an improvement in the knowledge ofA vinelandiialginate biosynthesis gene regulation in alginate productionprocesses in order to generate a tailored and affordable algi-nate product

6 Conclusion

In the present review we discuss that oxygen availabilityduring Azotobacter vinelandii cultures might exert a tight

control over the expression of alginate-related genes whichwill impact the quality of the polysaccharide or will regulateenzymatic activities that modified the nascent alginate chainCurrent evidence indicates a prevailing equilibrium in alg8and algL gene expression which is being regulated by oxygenavailability This equilibrium will further impact the alginatemolecular weight Accordingly more information regard-ing oxygen sensing transportation and signaling pathwaysduring specific culture conditions of A vinelandii will beneeded in order to obtain alginates with specific characteris-tics

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by a Grant from CONICYT-Chile(Project PCCI40039) and DI-PUCV 037-98 The authors


acknowledge Dr Nuno Rodrigues Dos Santos for his criticalcomments on the paper

References

[1] B H A Rehm ldquoBacterial polymers biosynthesis modificationsand applicationsrdquoNature ReviewsMicrobiology vol 8 no 8 pp578ndash592 2010

[2] S Bengtsson A R Pisco M A M Reis and P C Lemos ldquoPro-duction of polyhydroxyalkanoates from fermented sugar canemolasses by amixed culture enriched in glycogen accumulatingorganismsrdquo Journal of Biotechnology vol 145 no 3 pp 253ndash2632010

[3] F Clementi ldquoAlginate production by Azotobacter vinelandiirdquoCritical Reviews in Biotechnology vol 17 no 4 pp 327ndash361 1997

[4] D E Pszczola ldquoDiscovering treasures of the deeprdquo Food Tech-nology vol 52 no 4 pp 74ndash80 1998

[5] I W Sutherland Biotechnology of Microbial Exopolysacchary-des Cambridge University Press Cambridge UK 1990

[6] U Remminghorst and B H A Rehm ldquoBacterial alginates frombiosynthesis to applicationsrdquo Biotechnology Letters vol 28 no21 pp 1701ndash1712 2006

[7] J L Geddie and I W Sutherland ldquoThe effect of acetylation oncation binding by algal and bacterial alginatesrdquo Biotechnologyand Applied Biochemistry vol 20 no 1 pp 117ndash129 1994

[8] T Castillo E Heinzle S Peifer K Schneider and C F PenaM ldquoOxygen supply strongly influences metabolic fluxes theproduction of poly(3-hydroxybutyrate) and alginate and thedegree of acetylation of alginate in Azotobacter vinelandiirdquo Pro-cess Biochemistry vol 48 no 7 pp 995ndash1003 2013

[9] C Flores S Moreno G Espın C Pena and E GalindoldquoExpression of alginases and alginate polymerase genes inresponse to oxygen and their relationship with the alginatemolecularweight inAzotobacter vinelandiirdquoEnzyme andMicro-bial Technology vol 53 no 2 pp 85ndash91 2013

[10] C Kıvılcımdan Moral O Dogan and F D Sanin ldquoEffect ofoxygen tension and medium components on monomer distri-bution of alginaterdquoApplied Biochemistry and Biotechnology vol176 no 3 pp 875ndash891 2015

[11] ADıaz-Barrera AAguirre J Berrios andFAcevedo ldquoContin-uous cultures for alginate production by Azotobacter vinelandiigrowing at different oxygen uptake ratesrdquo Process Biochemistryvol 46 no 9 pp 1879ndash1883 2011

[12] A Dıaz-Barrera E Soto and C Altamirano ldquoAlginate pro-duction and alg8 gene expression by Azotobacter vinelandiiin continuous culturesrdquo Journal of Industrial Microbiology andBiotechnology vol 39 no 4 pp 613ndash621 2012

[13] A Dıaz-Barrera F Martınez F Guevara Pezoa F Acevedo andB Lin ldquoEvaluation of gene expression and alginate productionin response to oxygen transfer in continuous culture of Azoto-bacter vinelandiirdquo PLoS ONE vol 9 no 8 Article ID e1059932014

[14] N Rhein-KnudsenM T Ale andA SMeyer ldquoSeaweed hydro-colloid production an update on enzyme assisted extractionandmodification technologiesrdquoMarine Drugs vol 13 no 6 pp3340ndash3359 2015

[15] W Sabra A-P Zeng and W-D Deckwer ldquoBacterial alginatephysiology product quality and process aspectsrdquoAppliedMicro-biology and Biotechnology vol 56 no 3-4 pp 315ndash325 2001

[16] B H A Rehm and S Valla ldquoBacterial alginates biosynthesisand applicationsrdquo Applied Microbiology and Biotechnology vol48 no 3 pp 281ndash288 1997

[17] P Gacesa ldquoBacterial alginate biosynthesismdashrecent progress andfuture prospectsrdquo Microbiology vol 144 no 5 pp 1133ndash11431998

[18] D Hoefer J K Schnepf T R Hammer M Fischer and CMarquardt ldquoBiotechnologically produced microbial alginatedressings show enhanced gel forming capacity compared tocommercial alginate dressings of marine originrdquo Journal ofMaterials Science Materials in Medicine vol 26 no 4 article162 2015

[19] E Ruvinov and S Cohen ldquoAlginate biomaterial for the treat-ment ofmyocardial infarction progress translational strategiesand clinical outlookrdquo Advanced Drug Delivery Reviews vol 96pp 54ndash76 2016

[20] J Venkatesan I Bhatnagar P Manivasagan K-H Kang andS-K Kim ldquoAlginate composites for bone tissue engineering areviewrdquo International Journal of Biological Macromolecules vol72 pp 269ndash281 2015

[21] I A Brownlee A Allen J P Pearson et al ldquoAlginate as a sourceof dietary fiberrdquo Critical Reviews in Food Science and Nutritionvol 45 no 6 pp 497ndash510 2005

[22] M G Jensen M Kristensen and A Astrup ldquoEffect of alginatesupplementation on weight loss in obese subjects completing a12-wk energy-restricted diet a randomized controlled trialrdquoTheAmerican Journal of Clinical Nutrition vol 96 no 1 pp 5ndash132012

[23] P W Dettmar V Strugala and J Craig Richardson ldquoThe keyrole alginates play in healthrdquo Food Hydrocolloids vol 25 no 2pp 263ndash266 2011

[24] M Liu LDaiH Shi S Xiong andC Zhou ldquoIn vitro evaluationof alginatehalloysite nanotube composite scaffolds for tissueengineeringrdquo Materials Science and Engineering C vol 49 pp700ndash712 2015

[25] N V Konda S Singh B A Simmons and D Klein-Marcuschamer ldquoAn investigation on the economic feasibility ofmacroalgae as a potential feedstock for biorefineriesrdquo BioEnergyResearch vol 8 no 3 pp 1046ndash1056 2015

[26] IWDavidson IW Sutherland andC J Lawson ldquoLocalizationof O-acetyl groups of bacterial alginaterdquo Journal of GeneralMicrobiology vol 98 no 2 pp 603ndash606 1977

[27] G Skjak-Braeligk S Paoletti and T Gianferrara ldquoSelective acety-lation of mannuronic acid residues in calcium alginate gelsrdquoCarbohydrate Research vol 185 no 1 pp 119ndash129 1989

[28] H Ertesvag ldquoAlginate-modifying enzymes biological roles andbiotechnological usesrdquo Frontiers in Microbiology vol 6 no 5232015

[29] H Ertesvag S Valla and G Skjak-Braeligk ldquoEnzymatic alginatemodificationrdquo in Alginates Biology and Applications B H ARehm Ed Microbiology Monographs pp 95ndash115 SpringerBerlin Germany 2009

[30] W Sabra A-P Zeng H Lunsdorf and W-D Deckwer ldquoEffectof oxygen on formation and structure of Azotobacter vinelandiialginate and its role in protecting nitrogenaserdquo Applied andEnvironmentalMicrobiology vol 66 no 9 pp 4037ndash4044 2000

[31] J C Setubal P dos Santos B S Goldman et al ldquoGenomesequence of Azotobacter vinelandii an obligate aerobe special-ized to support diverse anaerobic metabolic processesrdquo Journalof Bacteriology vol 191 no 14 pp 4534ndash4545 2009

[32] E Post D Kleiner and J Oelze ldquoWhole cell respiration andnitrogenase activities in Azotobacter vinelandii growing in oxy-gen controlled continuous culturerdquo Archives of Microbiologyvol 134 no 1 pp 68ndash72 1983


[33] H L Sadoff ldquoEncystment and germination inAzotobacter vine-landiirdquo Bacteriological Reviews vol 39 no 4 pp 516ndash539 1975

[34] D Segura C Nunez and G Espın ldquoAzotobacter cystsrdquo inEncyclopedia of Life Sciences JohnWiley amp Sons New York NYUSA 2001

[35] A Dıaz-Barrera and E Soto ldquoBiotechnological uses of Azoto-bacter vinelandii current state limits and prospectsrdquo AfricanJournal of Biotechnology vol 9 no 33 pp 5240ndash5250 2010

[36] C Flores A Dıaz-Barrera FMartınez E Galindo andC PenaldquoRole of oxygen in the polymerization and de-polymerizationof alginate produced by Azotobacter vinelandiirdquo Journal ofChemical Technology and Biotechnology vol 90 no 3 pp 356ndash365 2015

[37] J Schmid V Sieber and B Rehm ldquoBacterial exopolysaccha-rides biosynthesis pathways and engineering strategiesrdquo Fron-tiers in Microbiology vol 6 2015

[38] J C Whitney and P L Howell ldquoSynthase-dependent exopoly-saccharide secretion in Gram-negative bacteriardquo Trends inMicrobiology vol 21 no 2 pp 63ndash72 2013

[39] I D Hay Z U Rehman A Ghafoor and B H A Rehm ldquoBac-terial biosynthesis of alginatesrdquo Journal of Chemical Technologyand Biotechnology vol 85 no 6 pp 752ndash759 2010

[40] E Galindo C Pena C Nunez D Segura andG Espın ldquoMolec-ular and bioengineering strategies to improve alginate andpolydydroxyalkanoate production by Azotobacter vinelandiirdquoMicrobial Cell Factories vol 6 article 7 2007

[41] P J Tatnell N J Russell and P Gacesa ldquoGDP-mannose dehy-drogenase is the key regulatory enzyme in alginate biosynthesisin Pseudomonas aeruginosa evidence from metabolite studiesrdquoMicrobiology vol 140 no 7 pp 1745ndash1754 1994

[42] Z U Rehman Y Wang M F Moradali I D Hay and BH A Rehm ldquoInsights into the assembly of the alginate bio-synthesis machinery in Pseudomonas aeruginosardquo Applied andEnvironmentalMicrobiology vol 79 no 10 pp 3264ndash3272 2013

[43] M Fata Moradali I Donati I M Sims S Ghods and B HRehm ldquoAlginate polymerization and modification are linked inPseudomonas aeruginosardquomBio vol 6 no 3 Article ID e00453-15 2015

[44] H Mejıa-Ruız J Guzman S Moreno G Soberon-Chavez andG Espın ldquoThe Azotobacter vinelandii alg8 and alg44 genes areessential for alginate synthesis and can be transcribed from analgD-independent promoterrdquoGene vol 199 no 1-2 pp 271ndash2771997

[45] H Mejıa-Ruız S Moreno J Guzman et al ldquoIsolation and cha-racterization of an Azotobacter vinelandii algK mutantrdquo FEMSMicrobiology Letters vol 156 no 1 pp 101ndash106 1997

[46] L M Riley J T Weadge P Baker et al ldquoStructural and func-tional characterization of Pseudomonas aeruginosa AlgX roleof Algx in alginate acetylationrdquo Journal of Biological Chemistryvol 288 no 31 pp 22299ndash22314 2013

[47] M J Franklin and D E Ohman ldquoMutant analysis and cellularlocalization of the AlgI AlgJ and AlgF proteins required for Oacetylation of alginate in Pseudomonas aeruginosardquo Journal ofBacteriology vol 184 no 11 pp 3000ndash3007 2002

[48] P Baker T Ricer P J Moynihan et al ldquoP aeruginosa SGNHhydrolase-like proteins AlgJ and AlgX have similar topologybut separate and distinct roles in alginate acetylationrdquo PLoSPathogens vol 10 no 8 Article ID e1004334 2014

[49] A Vazquez S Moreno J Guzman A Alvarado and G EspınldquoTranscriptional organization of the Azotobacter vinelandiialgGXLVIFA genes characterization of algFmutantsrdquoGene vol232 no 2 pp 217ndash222 1999

[50] B I G Svanem W I Strand H Ertesvag et al ldquoThe catalyticactivities of the bifunctional Azotobacter vinelandii mannuro-nan C-5-epimerase and alginate lyase AlgE7 probably originatefrom the same active site in the enzymerdquo Journal of BiologicalChemistry vol 276 no 34 pp 31542ndash31550 2001

[51] M Gimmestad H Ertesvag T M B Heggeset O AarstadB I G Svanem and S Valla ldquoCharacterization of three newAzotobacter vinelandii alginate lyases one of which is involvedin cyst germinationrdquo Journal of Bacteriology vol 191 no 15 pp4845ndash4853 2009

[52] S Jain and D E Ohman ldquoRole of an alginate lyase for alginatetransport in mucoid Pseudomonas aeruginosardquo Infection andImmunity vol 73 no 10 pp 6429ndash6436 2005

[53] H Ertesvag H K Hoslashidal I K Hals A Rian B Doseth and SValla ldquoA family of modular type mannuronan C-5-epimerasegenes controls alginate structure in Azotobacter vinelandiirdquoMolecular Microbiology vol 16 no 4 pp 719ndash731 1995

[54] C Nunez R Leon J Guzman G Espın and G Soberon-Chavez ldquoRole of Azotobacter vinelandii mucA and mucC geneproducts in alginate productionrdquo Journal of Bacteriology vol182 no 23 pp 6550ndash6556 2000

[55] L Lloret R Barreto R Leon et al ldquoGenetic analysis of thetranscriptional arrangement of Azotobacter vinelandii alginatebiosynthetic genes identification of two independent promot-ersrdquoMolecular Microbiology vol 21 no 3 pp 449ndash457 1996

[56] G Gaona C Nunez J B Goldberg et al ldquoCharacterization ofthe Azotobacter vinelandii algC gene involved in alginate andlipopolysaccharide productionrdquo FEMS Microbiology Lettersvol 238 no 1 pp 199ndash206 2004

[57] S Moreno R Najera J Guzman G Soberon-Chavez andG Espın ldquoRole of alternative 120590 factor AlgU in encystment ofAzotobacter vinelandiirdquo Journal of Bacteriology vol 180 no 10pp 2766ndash2769 1998

[58] M Castaneda J Sanchez S Moreno C Nunez and G EspınldquoThe global regulators GacA and 120590119878 form part of a cascade thatcontrols alginate production in Azotobacter vinelandiirdquo Journalof Bacteriology vol 183 no 23 pp 6787ndash6793 2001

[59] J Manzo M Cocotl-Yanez T Tzontecomani et al ldquoPost-transcriptional regulation of the alginate biosynthetic gene algDby the GacRsm system in Azotobacter vinelandiirdquo Journal ofMolecular Microbiology and Biotechnology vol 21 no 3-4 pp147ndash159 2012

[60] M A Azevedo A I Bourbon A A Vicente and M ACerqueira ldquoAlginatechitosan nanoparticles for encapsulationand controlled release of vitamin B

2rdquo International Journal of

Biological Macromolecules vol 71 pp 141ndash146 2014[61] X Zhao B Li C Xue and L Sun ldquoEffect of molecular weight

on the antioxidant property of low molecular weight alginatefrom Laminaria japonicardquo Journal of Applied Phycology vol 24no 2 pp 295ndash300 2012

[62] C A Bonino M D Krebs C D Saquing et al ldquoElectro-spinning alginate-based nanofibers from blends to crosslinkedlow molecular weight alginate-only systemsrdquo CarbohydratePolymers vol 85 no 1 pp 111ndash119 2011

[63] K Fujiki H Matsuyama and T Yano ldquoProtective effect ofsodium alginates against bacterial infection in common carpCyprinus carpio Lrdquo Journal of Fish Diseases vol 17 no 4 pp349ndash355 1994

[64] T Kuda H Goto M Yokoyama and T Fujii ldquoEffects of dietaryconcentration of laminaran and depolymerised alginate on ratcecalmicroflora and plasma lipidsrdquo Fisheries Science vol 64 no4 pp 589ndash593 1998


[65] T Kuda T Yano N Matsuda and M Nishizawa ldquoInhibitoryeffects of laminaran and low molecular alginate against theputrefactive compounds produced by intestinal microflora invitro and in ratsrdquo Food Chemistry vol 91 no 4 pp 745ndash7492005

[66] I Pajic-Lijakovic S Levic M Hadnađev et al ldquoStructuralchanges of Ca-alginate beads caused by immobilized yeast cellgrowthrdquo Biochemical Engineering Journal vol 103 pp 32ndash382015

[67] F E Vasile A M Romero M A Judis and M F MazzobreldquoProsopis alba exudate gum as excipient for improving fish oilstability in alginatemdashchitosan beadsrdquo Food Chemistry vol 190pp 1093ndash1101 2016

[68] F Mancini L Montanari D Peressini and P FantozzildquoInfluence of alginate concentration and molecular weight onfunctional properties of mayonnaiserdquo LWTmdashFood Science andTechnology vol 35 no 6 pp 517ndash525 2002

[69] O Aizpurua-Olaizola P Navarro A Vallejo M Olivares NEtxebarria and A Usobiaga ldquoMicroencapsulation and storagestability of polyphenols from Vitis vinifera grape wastesrdquo FoodChemistry vol 190 pp 614ndash621 2016

[70] W Cheng C-H Liu C-M Kuo and J-C Chen ldquoDietaryadministration of sodium alginate enhances the immune abilityof white shrimp Litopenaeus vannamei and its resistance againstVibrio alginolyticusrdquo Fish and Shellfish Immunology vol 18 no1 pp 1ndash12 2005

[71] M D Wilcox I A Brownlee J C Richardson P W Dettmarand J P Pearson ldquoThe modulation of pancreatic lipase activityby alginatesrdquo Food Chemistry vol 146 pp 479ndash484 2014

[72] B An H Lee S Lee S Lee and J Choi ldquoDetermining theselectivity of divalent metal cations for the carboxyl group ofalginate hydrogel beads during competitive sorptionrdquo Journalof Hazardous Materials vol 298 pp 11ndash18 2015

[73] W Cheng R-T Tsai and C-C Chang ldquoDietary sodiumalginate administration enhances Mx gene expression of thetiger grouper Epinephelus fuscoguttatus receiving poly ICrdquoAquaculture vol 324-325 pp 201ndash208 2012

[74] S-T Chiu R-T Tsai J-P Hsu C-H Liu and W ChengldquoDietary sodium alginate administration to enhance the non-specific immune responses and disease resistance of the juve-nile grouper Epinephelus fuscoguttatusrdquo Aquaculture vol 277no 1-2 pp 66ndash72 2008

[75] C-H Liu S-P Yeh C-M Kuo W Cheng and C-H ChouldquoThe effect of sodium alginate on the immune response oftiger shrimp via dietary administration activity and gene trans-criptionrdquo Fish and Shellfish Immunology vol 21 no 4 pp 442ndash452 2006

[76] K Fujiki and T Yano ldquoEffects of sodium alginate on the non-specific defence system of the common carp (Cyprinus carpioL)rdquo Fish and Shellfish Immunology vol 7 no 6 pp 417ndash4271997

[77] H Tomida T Yasufuku T Fujii Y Kondo T Kai and MAnraku ldquoPolysaccharides as potential antioxidative compoundsfor extended-releasematrix tabletsrdquoCarbohydrate Research vol345 no 1 pp 82ndash86 2010

[78] L L Oglesby S Jain and D E Ohman ldquoMembrane topologyand roles ofPseudomonas aeruginosaAlg8 andAlg44 in alginatepolymerizationrdquo Microbiology vol 154 no 6 pp 1605ndash16152008

[79] I M Saxena R M Jr Brown M Fevre R A Geremia and BHenrissat ldquoMultidomain architecture of 120573-glycosil tranferases

implications for mechanism of actionrdquo Journal of Bacteriologyvol 177 no 6 pp 1419ndash1419 1995

[80] U Remminghorst and B H A Rehm ldquoIn vitro alginate poly-merization and the functional role of Alg8 in alginate produc-tion by Pseudomonas aeruginosardquo Applied and EnvironmentalMicrobiology vol 72 no 1 pp 298ndash305 2006

[81] MMerighi V T LeeMHyodo YHayakawa and S Lory ldquoThesecond messenger bis-(31015840-51015840)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesisin Pseudomonas aeruginosardquo Molecular Microbiology vol 65no 4 pp 876ndash895 2007

[82] S Alexeeva K J Hellingwerf and M J Teixeira de MattosldquoQuantitative assessment of oxygen availability perceived aero-biosis and its effect on flux distribution in the respiratory chainof Escherichia colirdquo Journal of Bacteriology vol 184 no 5 pp1402ndash1406 2002

[83] J Oelze ldquoRespiratory protection of nitrogenase in Azotobacterspecies Is a widely held hypothesis unequivocally supported byexperimental evidencerdquo FEMS Microbiology Reviews vol 24no 4 pp 321ndash333 2000

[84] M A Trujillo-Roldan S Moreno D Segura E Galindo andG Espın ldquoAlginate production by an Azotobacter vinelandiimutant unable to produce alginate lyaserdquo Applied Microbiologyand Biotechnology vol 60 no 6 pp 733ndash737 2003

[85] M A Trujillo-Roldan S Moreno G Espın and E GalindoldquoThe roles of oxygen and alginate-lyase in determining themolecular weight of alginate produced by Azotobacter vinel-andiirdquo Applied Microbiology and Biotechnology vol 63 no 6pp 742ndash747 2004

[86] A Dıaz-Barrera C Pena and E Galindo ldquoThe oxygen transferrate influences the molecular mass of the alginate produced byAzotobacter vinelandiirdquo Applied Microbiology and Biotechnol-ogy vol 76 no 4 pp 903ndash910 2007

[87] A Dıaz-Barrera P Silva R Avalos and F Acevedo ldquoAlginatemolecular mass produced byAzotobacter vinelandii in responseto changes of the O

2transfer rate in chemostat culturesrdquo Bio-

technology Letters vol 31 no 6 pp 825ndash829 2009[88] E Lozano E Galindo and C F Pena ldquoOxygen transfer rate

during the production of alginate by Azotobacter vinelandiiunder oxygen-limited and non oxygen-limited conditionsrdquoMicrobial Cell Factories vol 10 article 13 2011

[89] C Pena M A Trujillo-Roldan and E Galindo ldquoInfluenceof dissolved oxygen tension and agitation speed on alginateproduction and its molecular weight in cultures of Azotobactervinelandiirdquo Enzyme and Microbial Technology vol 27 no 6 pp390ndash398 2000

[90] J Green and M S Paget ldquoBacterial redox sensorsrdquo NatureReviews Microbiology vol 2 no 12 pp 954ndash966 2004

[91] G Wu A J G Moir G Sawers S Hill and R K Poole ldquoBio-synthesis of poly-120573-hydroxybutyrate (PHB) is controlled byCydR (Fnr) in the obligate aerobe Azotobacter vinelandiirdquoFEMS Microbiology Letters vol 194 no 2 pp 215ndash220 2001

[92] A Dıaz-Barrera R Andler I Martınez and C Pena ldquoPoly-3-hydroxybutyrate production by Azotobacter vinelandii strainsin batch cultures at different oxygen transfer ratesrdquo Journal ofChemical Technology amp Biotechnology 2015

[93] J M Martınez-Salazar S Moreno R Najera et al ldquoCharac-terization of the genes coding for the putative sigma factorAlgU and its regulators MucA MucB MucC and MucD inAzotobacter vinelandii and evaluation of their roles in alginatebiosynthesisrdquo Journal of Bacteriology vol 178 no 7 pp 1800ndash1808 1996


[94] R Leon and G Espın ldquoflhDC but not fleQ regulates flagellabiogenesis in Azotobacter vinelandii and is under AlgU andCydR negative controlrdquo Microbiology vol 154 no 6 pp 1719ndash1728 2008

[95] C Nunez A V Bogachev G Guzman I Tello J Guzman andG Espın ldquoThe Na+-translocating NADH ubiquinone oxido-reductase ofAzotobacter vinelandii negatively regulates alginatesynthesisrdquoMicrobiology vol 155 no 1 pp 249ndash256 2009

[96] Y V Bertsova A V Bogachev and V P Skulachev ldquoNon-coupled NADH ubiquinone oxidoreductase of Azotobactervinelandii is required for diazotrophic growth at high oxygenconcentrationsrdquo Journal of Bacteriology vol 183 no 23 pp6869ndash6874 2001

[97] M Bekker S Alexeeva W Laan G Sawers J T De Mattosand K Hellingwerf ldquoThe ArcBA two-component system ofEscherichia coli is regulated by the redox state of both theubiquinone and themenaquinone poolrdquo Journal of Bacteriologyvol 192 no 3 pp 746ndash754 2010

[98] D Georgellis O Kwon and E C C Lin ldquoQuinones as the redoxsignal for the Arc two-component system of bacteriardquo Sciencevol 292 no 5525 pp 2314ndash2316 2001

[99] R Malpica G R Pena Sandoval C Rodrıguez B Franco andD Georgellis ldquoSignaling by the Arc two-component systemprovides a link between the redox state of the quinone pool andgene expressionrdquo Antioxidants and Redox Signaling vol 8 no5-6 pp 781ndash795 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


2000

1500

1000

500

0

Num

ber o

f pub

licat

ions

1975 1995 2015

Year(a)

1657 biochemistry genetics and molecular biology1093 medicine1083 materials science1057 chemical engineering943 chemistry900 engineering769 agricultural and biological sciences748 immunology and microbiology604 pharmacology toxicology and pharmaceutics1147 others

Total = 43600

(b)

Figure 1 (a) Number of publications indexed in Scopus database(August 2015) using keyword alginate (minus) in title abstract orkeywords (b) Percentage of theword alginatedistributed in differentsubject areas

molecular mechanism underlying polymer biosynthesis inrelationship with the oxygen availability during the fermen-tation process

2 Alginate Structure Chemical Structureand Applications

Over the past 40 years a growing interest in the use of alginatehas been observed including different areas ranging fromgenetics to pharmaceutics (Figure 1)

Alginate has been placed as the second biopolymerderived from seaweeds with greater demand in the

Table 1 Summary of biotechnological and pharmaceutical applica-tions of alginates based on their molecular weights

Application of alginateMolecularweight(kDa)

Reference

Delivery of bioactive compoundsAntioxidantIn vivo tissue scaffoldsAntibacterialDietary supplementCell immobilization

asymp15ndash120 [60ndash66]

Food stabilizer and preservingagentMicroencapsulation and storagestabilityAntibacterialBioremediationWound healing

asymp120ndash290 [62 63 67ndash76]

Modulation of enzymatic activityExtended-release tabletcompound

500ndash941 [71 77]

hydrocolloidsrsquo industry [14] Currently the only economicway to obtain commercial alginate used for most applicationsis through its extraction frommarine algae the cost of whichranges between US$ 2 and 20kg and with a total marketvalue of around US$ 339 million [14] Furthermore alginatesof very high purity are used in the pharmaceutical industrywhere they are sold for up to US$ 3200kg

Since alginate is a biodegradable and a biocompatiblepolysaccharide it presents a panoply of food pharmaceu-tical and biotechnological applications (Figure 1(b)) In thefood and pharmaceutical industries alginate is mainly usedas a stabilizing thickening or gel-film-forming agent [615ndash17] Table 1 in medicine it is used as wound healingmaterial [18] as part of medical treatments [19 20] or asdietary fiber supplements [21 22] Alginate showed potentialbeneficial physiological effects in the gastrointestinal tract[23] Moreover hydrogel-alginates are being investigated inbiotechnology as drug delivery agents as cell encapsulationmaterial and as scaffold material in tissue engineering [24]

Alginate is the main structural component of brownmarine algae (Laminaria andMacrocystis) representing about32 of dry biomass [25] consisting in variable amounts ofM- G- and MG-residues linked by 1rarr4 glycosidic bonds[7] On the other hand alginates produced by bacteria aresubmitted to esterification with O-acetyl groups at the O-2 andor O-3 of the M-residues [26] where the majority oftheM-residues aremono-O-acetylated and infrequentlywith23-di-acetylated [27] (Figure 2) Because the monomericchemical structure of bacterial alginate and the sequencelength determine the mechanical properties of the alginatesone of the aims of different investigations is the possibility ofmanipulating the composition alginates for specific applica-tions have been intensively investigated [28 29]

The obligate aerobe bacterium Azotobacter vinelandiiproduces alginate that acts as a diffusion barrier for nutrientsand oxygen [30 31] It was reported as a bacterium with a


O

OO

OO

OO

OO

O OO

O

OH

OH

OHOH

HO

OO

M M M

OH

OHG G

OHOH

OH

G

minusOOC

minusOOC

minusOOC

minusOOC

minusOOC

minusOOCH3C

CH3

CH3

C=O

C=OC=O















OM

Perip

lasm

Cyto

plas

mIM

P

PG


PP

P

PP

PAlgA

AlgA

AlgC

AlgD

GDP

AlgVgAlgFF

AAlgXA

AlgJ


AlyB

E1 E2E6

E4E5

E3

AlyA3 AlgE7

AlyA2

AlyA1AlgF



Acetyl donor

Acetyl group

OM outer membrane

PP

PGDP





gAlg44

AlgX


AlgL

AlgK

Alg44AlgG


c-di-GMP

GMP + Pi

Ca2+

Ca2+

Ca2+Ca2+ Ca2+














alyA3 alyBalyA1


OM

PGPe

ripla

sm

Alginate

IMCy

topl

asm

MucB

MucA

AlgU

GacS

GacA GacA

algCrpoS

algDrsmA

rsmrsmZ1rsmZ2

malgDalgU

120590D

120590S

ADP ATP

P

Cell wall stress

5998400UTR














2




2


2


2



2





2









2


2






2







High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM



NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+









6 Conclusion





Acknowledgments




References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




O

OO

OO

OO

OO

O OO

O

OH

OH

OHOH

HO

OO

M M M

OH

OHG G

OHOH

OH

G

minusOOC

minusOOC

minusOOC

minusOOC

minusOOC

minusOOCH3C

CH3

CH3

C=O

C=OC=O















OM

Perip

lasm

Cyto

plas

mIM

P

PG


PP

P

PP

PAlgA

AlgA

AlgC

AlgD

GDP

AlgVgAlgFF

AAlgXA

AlgJ


AlyB

E1 E2E6

E4E5

E3

AlyA3 AlgE7

AlyA2

AlyA1AlgF



Acetyl donor

Acetyl group

OM outer membrane

PP

PGDP





gAlg44

AlgX


AlgL

AlgK

Alg44AlgG


c-di-GMP

GMP + Pi

Ca2+

Ca2+

Ca2+Ca2+ Ca2+














alyA3 alyBalyA1


OM

PGPe

ripla

sm

Alginate

IMCy

topl

asm

MucB

MucA

AlgU

GacS

GacA GacA

algCrpoS

algDrsmA

rsmrsmZ1rsmZ2

malgDalgU

120590D

120590S

ADP ATP

P

Cell wall stress

5998400UTR














2




2


2


2



2





2









2


2






2







High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM



NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+









6 Conclusion





Acknowledgments




References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




OM

Perip

lasm

Cyto

plas

mIM

P

PG


PP

P

PP

PAlgA

AlgA

AlgC

AlgD

GDP

AlgVgAlgFF

AAlgXA

AlgJ


AlyB

E1 E2E6

E4E5

E3

AlyA3 AlgE7

AlyA2

AlyA1AlgF



Acetyl donor

Acetyl group

OM outer membrane

PP

PGDP





gAlg44

AlgX


AlgL

AlgK

Alg44AlgG


c-di-GMP

GMP + Pi

Ca2+

Ca2+

Ca2+Ca2+ Ca2+














alyA3 alyBalyA1


OM

PGPe

ripla

sm

Alginate

IMCy

topl

asm

MucB

MucA

AlgU

GacS

GacA GacA

algCrpoS

algDrsmA

rsmrsmZ1rsmZ2

malgDalgU

120590D

120590S

ADP ATP

P

Cell wall stress

5998400UTR














2




2


2


2



2





2









2


2






2







High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM



NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+









6 Conclusion





Acknowledgments




References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







alyA3 alyBalyA1


OM

PGPe

ripla

sm

Alginate

IMCy

topl

asm

MucB

MucA

AlgU

GacS

GacA GacA

algCrpoS

algDrsmA

rsmrsmZ1rsmZ2

malgDalgU

120590D

120590S

ADP ATP

P

Cell wall stress

5998400UTR














2




2


2


2



2





2









2


2






2







High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM



NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+









6 Conclusion





Acknowledgments




References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








2




2


2


2



2





2









2


2






2







High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM



NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+









6 Conclusion





Acknowledgments




References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







2


2






2







High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM



NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+









6 Conclusion





Acknowledgments




References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




High O2

O2

O2 O2

O2 O2

O2

O2

O2 O2 O2

O2 O2O2

O2

O2

O2 O2O2

O2 O2

O2 O2

Low O2

Q8H2

Na+NQR

NADH NADH NAD+

PGO

M

Alginate

Perip

lasm

Na+ Na+Na+

Na+Na+

B B

P

P

P

A A

algC algC

algD

A A

Cytochromeoxidase

Cyto

plas

m

Na+ Na+Na+

Na+Na+

IM



NADHdehydrogenase

CytochromeoxidaseQ8

BB

A

P

A

alg8 alg44

NAD+









6 Conclusion





Acknowledgments




References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





References



















































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





















































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



review article alginate biosynthesis in azotobacter...

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